Project Summary Rheumatoid arthritis (RA) is a common chronic inflammatory disease that results in joint swelling and pain, radiographic damage, and disability. Anti-citrullinated protein antibodies (ACPA) are ~95% specific for RA. Citrullination, a post-translational process of deimination of arginine residues, is catalyzed by peptidylarginine deiminases (PADs) such as PAD4. A variety of proteins are citrullinated in RA, and PAD4 is thought to contribute to RA pathogenesis by providing a continual source of citrullinated autoantigens that induce autoimmune responses. Dr. Darrah and colleagues identified antibodies (Abs) to PAD4 itself, which are associated with: (i) disease severity, (ii) serum ACPA, and (iii) severe erosive joint damage. They also discovered a subset of anti-PAD4 Abs cross-reactive with PAD3. These unexpectedly cause increased PAD4 enzymatic activity and their presence in serum defines a subset of RA patients exhibiting the most erosive damage. There are many unanswered questions regarding B cell/antibody immune responses to PAD4 and PAD3 in RA. These questions will be addressed using a registry/repository of well-phenotyped RA patients (mostly Caucasian) and access to a large database and repository of African-Americans with RA. Novel cutting-edge techniques will be used to study B cell immune responses at the individual cell level, including sequencing of paired heavy and light chain antibodies from serum and transcripts from circulating plasmablasts/B cells. We will define molecular characteristics of anti-PAD Abs (specificity, epitope mapping, etc.). In tandem with the functional studies above, structural studies will be used define the molecular determinants of immune complexes comprising PAD4 antigen bound to anti-PAD4 or anti-PAD4/3 mAbs (Fab variants). Thus, the cellular, molecular, biochemical, and structural mechanisms of antibody-mediated effects on PAD4 will be elucidated. Our Specific Aims are: 1. To identify and quantify the molecular species of anti- PAD IgG and IgA antibodies circulating in serum of RA patients and to identify the B-cell subset of origin. We will characterize anti-PAD4 and PAD4/3 IgG and IgA antibody protein repertoires in RA patients and perform Next-Gen sequencing of key B lineage subsets. This will allow us to identify, quantify, and rank by abundance and subclass all Ig clonotypes in the anti-PAD repertoires. 2. To define the molecular correlates of anti-PAD binding and modulation of PAD4 activity. a. We will define the molecular characteristics of monoclonal PAD antibodies that are required for PAD4 binding, cross-reactivity with PAD3, and activation of PAD4 catalysis. Using serum of RA patients, we will directly define the Ig class and subclass repertoire of polyclonal circulating anti-PAD antibodies in RA serum and determine how these may correlate with clinical characteristics. 3. To determine the structure of PAD-containing immune complexes and the mechanisms by which anti-PAD4/3 cross-reactive antibodies mediate PAD4 activation. Structural studies will define the molecular determinants of immune complexes comprising PAD4 antigen bound to anti-PAD4 or anti-PAD4/3 mAbs (Fab variants).